UV and chemical mutagenesis in Escherichia coli results from a specialized mechanism of translesion synthesis. This process specifically requires the umuDC gene products, whose expression and activity is controlled by the SOS regulatory system, with UmuD becoming activated to UmuD' by a proteolytic cleavage mediated by activated RecA. It also requires the RecA protein and DNA polymerase III. The long term goals of this research are to 1) gain a detailed understanding of the molecular mechanism responsible for UV and chemical mutagenesis in E. coli with a particular emphasis on elucidating the roles of the umuDC gene products in this process, 2) investigate possible physiological roles of the umuDC gene products in processes beside translesion synthesis such as cell cycle regulation, and 3) use insights gained from these studies to understand the basis of UV and chemical mutagenesis in higher organisms including humans. Insights into the mechanism of this type of mutagenesis will have important implications for understanding the molecular basis of chemical carcinogenesis and genetic diseases. The three dimensional structure of UmuD'2, UmuD2 and UmuD'-UmuD will be determined by NMR in collaboration with Dr. Wagner's laboratory. A set of monocysteine derivatives of UmuD and UmuD' has been constructed and the presence of the single thiol group in each protein will be exploited in a variety of ways to learn more about the structure and interactions of UmuD and UmuD'. The nature of the interaction between UmuD and RecA* that leads to UmuD cleavage will be investigated and the key regions of UmuD and RecA involved in this interaction will be identified. A variety of techniques will be used to investigate the nature of the interactions of UmuD and UmuD' with UmuC and the nature of the interactions of UmuD' and UmuC with DNA polymerase III and with RecA*. Analyses of translesion synthesis will be continued using UmuD' and UmuC-His6 purified without the use of denaturing agents. Various experimental strategies will be utilized to investigate the mechanism of UmuD'/UmuC dependent translesion synthesis with special emphasis on the roles of the UmuD' and UmuC proteins and their possible interactions with particular subunits of DNA polymerase III holoenzyme. Experiments will be carried out to investigate the roles of the umuDC gene products in cell cycle regulation and in replication restart. On the longer term, knowledge gained from these studies should help us understand the function of apparent eukaryotic homologs of UmuC.